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\title{Quantitative Outcomes of a One Health Approach to Investigate the First Outbreak of African Swine Fever in the Republic of}
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             \author[1]{Raoul Emeric Guetiya  Wadoum}

             \author[2]{Jacqueline Kasiiti  Lichoti}

             \author[3]{Nantima  Noelina}

             \author[4]{Bitek  Austine}

             \author[5]{Leno  Amara}

             \author[6]{Abdul K.  Sesay}

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\date{\small \em Received: 9 December 2019 Accepted: 4 January 2020 Published: 15 January 2020}

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\begin{abstract}
        


African swine fever (ASF) outbreaks have been reported in Sub-Saharan countries, including West Africa states, but has never been notified in the Republic of Sierra Leone. This is the first report describing field epidemiological and laboratory investigations into the outbreak of fatal pig disease in western rural and urban districts, Freetown. A preliminary finding indicated that pigs exhibited clinical and necropsy signs suggestive of ASF. Serological (ELISA) and molecular (qRT-PCR) methods used to confirm and investigate the outbreak yielded three positive results for the ASF antibody and all negative for Swine flu; thus, confirming ASF as the etiology agent. 

\end{abstract}


\keywords{one health; zoonotic diseases; african swine fever virus; influenza a viruses; sierra leone.}

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\section[{Introduction}]{Introduction}\par
frican swine fever (ASF) is a significant disease of domestic pigs caused by a DNA arbovirus virus belonging to the family Asfaviridae \hyperref[b0]{(Dixon et al., 2005)}. To date, there are 22 different genotypes described based on the p72 sequences, all of which circulate on the Africa continent \hyperref[b1]{(Boshoff et al., 2007)}. Due to its devastating economic impact in affected countries, the World Organization for Animal Health (OIE) listed ASF as a notifiable disease.\par
Outbreaks of ASF have been reported in Sub-Saharan countries, including West Africa states particularly in Benin, Burkina Faso, Cote d'Ivoire, the Gambia, Ghana, Guinea, Guinea-Bissau, Mali, Nigeria, Senegal, and Togo; however, the Republic of Sierra Leone never reported an epidemic of ASF \hyperref[b2]{[Bastos et al., 2003;}\hyperref[b5]{Brown et al., 2018;}\hyperref[b4]{Penrith et al., 2013]}. Nonetheless, the Ministry of Agriculture and Forestry (MAF), together with the Ministry of Health and Sanitation (MoHS) jointly announced on 23 October 2019 that an outbreak of an unknown disease was killing pigs in western rural and urban districts, Freetown. Preliminary investigation jointly conducted by MAF and the Food and Agriculture Organization of the United Nations (FAO) in the affected areas, indicated that pigs exhibited clinical signs matching suspect case definition for ASF as defined by OIE which included; high fever, depression, loss of appetite, hyperemia and cyanosis (seen as reddening) of the skin, particularly the ears and snout and eventually death.\par
Recognizing that pigs host various zoonotic pathogens, One Health (OH) approach aligned with global recommendation was established and implemented to investigate the etiological agents responsible for the epidemic, and institute prevention and control measures. OH approach promoted interdisciplinary collaboration and coordination, bringing A together health scientists and practitioners at the human, animal, and environment to strengthen emerging and re-emerging infectious disease surveillance and outbreak response.\par
Consequently, a OH rapid response team composed of epidemiologists from MAF, MoHS, FAO, and scientists from the University of Makeni, Njala University and CDC/AFFENET/FELTP intermediate students was deployed in affected communities to gather epidemiological information (morbidity, mortality and cases fatality rates, risk factors) in affected and unaffected farms, collect biological samples for laboratory confirmation and institute prevention and control measures to contain the outbreak.\par
Here we report preliminary field epidemiological and laboratory investigations findings into the first outbreak of ASF in the Republic of Sierra Leone. 
\section[{II.}]{II.} 
\section[{Materials and Methods}]{Materials and Methods} 
\section[{a) Localization of the study}]{a) Localization of the study}\par
Investigations were carried out from 29 October to 1 November 2019 by three sub teams derived from the One Health rapid response team. Each team was assigned communities where reports of death pigs had occurred to undertake active disease searches to identify cases using case definition for ASF provided by FAO. The case definition used was any pig herd with one or more age groups affected with high fever, depression, and loss of appetite, hyperemia, and cyanosis of the skin, particularly at the ears and snout and eventually death within 2-10 days in the affected areas from 10 th September 2019 to present.\par
Areas visited included Monkey Bush, Campbell Town, Samuel Town in Waterloo, Maburieh, Bengumah, Ibo Town, Bomeh in waterloo, Bomeh/Kingtom, Kroobay, Susan Bay, Moawharf and Racecourse Clintown (Figure \hyperref[fig_0]{1}). Each investigation team received forms/questionnaires for collecting epidemiological and laboratory data, and case definition checklist to assist them to identify cases. 
\section[{b) Sample Collection}]{b) Sample Collection}\par
Infected and non-infected farms were visited for inspection and specimen recovered from more than 60 pigs of ages between 4 -12 months. Moreover, each team recorded GPS coordinate of various communities visited during the five days of field activities. For each pig, oral pharyngeal swabs, nasal mucosae swabs were collected using sterile swab collection kits and placed in 2 mL cryovial containing RNAlater (Ambion Inc., Austin, TX, USA). Cardiac puncture method was used for blood sample collection into a vacutainer tube with or without containing EDTA and plasma or serum recovered and stored until analysis. Fecal samples and tissues (spleen, liver, lymph nodes, lungs, heart, and kidneys) from dead pigs at postmortem were also collected.\par
During field expedition, scientists kept all samples on dry ice, and upon returning to the laboratory they were transferred to a -20 °C freezer then -80 °C until further analysis.\par
All specimens were collected in duplicate and transported to Central Veterinary Laboratory (CVL), Teko Makeni for testing for ASF, and to the University of Makeni Infectious Diseases laboratory (IDRL) for Swine Flu and other potential zoonotic diseases analysis. 
\section[{c) Serology assay for detection of ASF}]{c) Serology assay for detection of ASF}\par
All serum and blood samples were analyzed using a Multi-antigen indirect ELISA kit for the detection of ASF antibodies against P32, P62, and P72 ASF antigens manufactured by ID Vet diagnostics (ID Vet, 2019). Briefly, samples were added to the antigen precoated plate and incubated at room temperature, after washing to eliminate excess serum, a specific conjugate was added and incubated. The plates were further washed, and upon addition of substrate and stop solution respectively, incubated and optical density read on a Multiskan Sky Microplate Spectrophotometer (ThermoFisher Scientific). 
\section[{d) Detection of Swine Flu}]{d) Detection of Swine Flu}\par
i. RNA Extraction and qRT-PCR RNA from oral swabs, nasal swabs, serum, and plasma was extracted using QiaAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instruction and material eluted in 60µL of AVE buffer and stored at -80 °C until needed. Quantitative reverse transcription real-time PCR (qRT-PCR) for the detection of Human Influenza A H1 and H3 viruses was performed on a Light Cycler® 96 Instrument (Roche, Life Science) using a Primer Design? genesig kit (Southampton, United Kingdom) following the manufacturer's instruction with cut-off Ct set at 38. The PCR amplification reaction was performed in a 20 µL reaction mix as follows; 50 °C for 600 s (reverse transcription) and 95 °C for 15 min (DNA polymerase activation), followed by 50 cycles at 95 °C for 10 s (denaturation), and finally 60 °C for 60 s (annealing and extension). Positive results obtained were characterized by a sigmoid curve, showing an initial, rapid, exponential increase in fluorescence signal followed by a plateau. Negative reactions did not show any increase in fluorescence signal. A standard curve was created automatically with the Light Cycler® software in each run by plotting the cycle threshold number against the copy numbers of each standard, and quantification of unknown samples were inferred from the regression line. 
\section[{e) Data Analysis}]{e) Data Analysis}\par
Microsoft excel was used for all analyses and animal-related factors characterizing animals or herd such as breed, age, herd size, grazing system, morbidity, mortality, and breeding system were established through univariate analysis.\par
Recent and past exposure to ASF was determined by the presence of ASF specific antibodies, and seroprevalence defined as the proportion of positive ASF IgG/IgM antibodies among the sampled pigs, and molecular assay data analysis performed on the LightCycler® 96 Application Software Version 1.1. 
\section[{III.}]{III.} 
\section[{Results and Discussion}]{Results and Discussion}\par
Pig production in Sierra Leone is mainly smallscale traditional using indigenous breeds of pigs, which are small, short, and sturdy with little hair covering on the skin. Some farmers keep exotic breeds, mainly large white race, and Duroc. There has been a lot of crossbreeding between the local and the exotic breeds, giving rise to hybrids. The country has an estimated pig population of 57,877 pigs representing 1\% of the total livestock population in the country (Table  {\ref 1}). Western Urban and Western areas have an estimated pig population of 4,343 and 6,603 respectively representing about 20\% of the total pig population in the country.\par
The OH teams visited 48 households in 12 communities in Monkey Bush, Campbell Town, Samuel Town in Waterloo, Maburieh, Bengumah, Ibo Town, Bomeh in waterloo, Bomeh/Kingtom, Kroobay, Susan Bay, Moawarf and Racecourse. The majority of the pig farmers visited were keeping pigs on the free-range system, and they were roaming and scavenging under very poor hygienic conditions. Also, the farmers were dumping dead pigs in the nearby streams/tributaries where other pigs were scavenging, which increased the risk for ASF spreading to others districts such as Port Loko and Tonkolili; thus, representing a major risk of contamination in those districts.\par
Studies have highlighted that indiscriminate disposal of pig viscera, waste materials, poor biosecurity measures and salvage sale of survivor and sick pigs without testing contribute to in-country maintenance and spread of ASF \hyperref[b7]{(Kabuuka et al., 2014;} {\ref Muhangi et al., 2015;}\hyperref[b10]{Nantima et al., 2015)}.\par
The estimated herd mortality in the sampled households ranged from 0 to 99.4\%. The mortality rate was highest in local communities within the Western Urban district compared to the Western Rural district (Table  {\ref 2}). Kroobay in Western Urban had the highest herd mortality rate of 99.4\% while Colvert and Benguima in Western Rural districts had 0\% herd mortalities. These mortality disparities are concordant with previous studies indicating that ASF mortality varies between 30-70\% for moderately virulent strains and 100\% in naïve herds of domesticated pigs \hyperref[b11]{(Spickler, 2019)}.\par
Three hundred and thirty-nine (339) specimens were collected during investigation including, whole blood, serum, oral swabs, nasal swabs, fecal samples, and organs. The samples collected included; whole blood, serum, nasal and oral pharyngeal swabs as well as fecal samples. A post-mortem was undertaken on dead pigs, and organs collected, including spleen, lymph nodes, kidneys, liver, and heart.\par
Among the samples collected during the investigation, ASF ELISA detected antibodies in three samples from sick pigs (2 URC serum and 1 UKT (blood and serum)), indicative of exposure to the ASF virus (Table  {\ref 3}). Currently, there is no vaccine against ASF; therefore, the presence of antibodies in sick pigs is a result of exposure to the ASF virus. The behavior of farmers quickly selling off sick pigs to mitigate economic impact compromised the detection of virus and antibodies. By the time the response team arrives for investigation, most of the farmers didn't have sick animals. Also, most pigs sampled may not have been exposed to ASF, reason for a large number of negative samples. Considering the epidemiologic features of communities where the massive death of pigs occurred, the most probable source of infection suggested was through virus-contaminated pork products. OIE terrestrial manual for diagnostics of ASF recommends that investigation of new outbreaks should include the detection of specific antibodies in serum or extracts of tissues (OIE, 2019). These results confirmed the initial suspicion of ASF being the etiology agent responsible for the outbreak.\par
Even though findings confirmed ASF as the leading etiology of the outbreak, concerns raised about pigs been co-infected with other zoonotic disease were legitimate given that pigs are known to be susceptible to zoonotic diseases such as Ebola virus disease (Reston ebolavirus) and Swine flu (CDC, 2019;  {\ref Nelson et al., 2019)}. Accordingly, a total of 204 RNA derived from oral swabs, nasal swabs, and serum/plasma were screened by qRT-PCR for the detection of Human Influenza A H1 and H3 gene. Results reveal that no amplification was produced in the no-template control reaction wells while positive control used as standard at various concentration upon amplification, yielded a typical sigmoidal curve; thus, validating the assay. However, all samples tested were negative, with the amplification curve failing to display sigmoidal curves as positive control. Presently, the laboratory diagnosis of influenza virus infection in pigs typically relied upon the detection of the virus in nasal swabs. Serology to detect antibodies is of low value for swine Influenza virus surveillance because vaccination against the disease rely on inactivated H1N1 and H3N2 vaccines, and current serologic tests do not differentiate between vaccinated and infected animals \hyperref[b15]{(Detmer et al., 2013)}. Therefore, virological assays are currently preferred over serology for surveillance. In the current study, Human influenza A virus H1 and H3 weren't detected in the various samples analyzed; thus, supporting that pigs weren't co-infected with the Swine flu virus. Nevertheless, the non-detection of these viruses was crucial as appropriate information was tailored and provided to decrease public fear of the zoonotic potential of the outbreak in Freetown. The intensive community sensitization by the OH teams was satisfactory as farmers were engaged about issues of implementing biosecurity measures and had to bury and burn dead pigs in some localities. Some farmers that still had few pigs surviving was advised on the benefit of isolating sick pigs even though most farmers reported to have lost all their pigs. Sierra Leone has never reported an outbreak of African swine fever. During the epidemiological investigations, some farmers revealed that they experienced an outbreak similar to this one about three years ago. It is possible that outbreaks have been occurring without being reported to Livestock and Veterinary Services Division of MAF, or no response was provided by the weak Veterinary service of Sierra Leone.\par
The major risk factors that could have exacerbated the spread of ASF during this outbreak were selling sick and dead pigs by the affected farmers, disposal of dead pigs in the streams/tributaries where other pigs were scavenging, and inadequate biosecurity measures. These could have been among the reasons why the disease was more severe in localities in Western Urban compared to Western Rural district.\par
There is no specific policy for controlling ASF in Sierra Leone. The control of ASF is governed under the Animal Diseases Ordinance (1948). The challenge is that most of the legislation is outdated and therefore lacks relevant provisions in controlling the disease. The old law is currently being revised, and hopefully, the new law will address ASF control. The country does not have a compensation policy to support farmers that have lost pigs to ASF. Also, there is no surveillance study related to ASF, and other pig diseases currently being carried out in the country.\par
IV. 
\section[{Conclusion}]{Conclusion}\par
Our findings confirmed ASF as the etiology of the reported outbreak of ASF in the republic of Sierra Leone. We demonstrated that rapid response and community engagement following the One Health approach is an effective means to alleviate fear and panic during an outbreak.  
\section[{Recommendations}]{Recommendations} 
\section[{Ethics Statement}]{Ethics Statement}\par
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this was the Sierra Leone government investigation into the outbreak. However, animal handling and sample collection respected the rules formulated under the Animal Welfare Act by the United States Department of Agriculture (USDA) and by adopting ARRIVE guidelines \hyperref[b16]{(Kilkenny et al., 2011)}. \begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Figure 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{} \par 
\begin{longtable}{P{0.85\textwidth}}
1. African Swine Fever is a notifiable disease in Sierra\\
Leone, and the Government should urgently notify\\
OIE and RECs about the outbreak\\
2. A program to train farmers on implementation of\\
biosecurity practices should immediately be\\
undertaken by Government\\
3. Adequate community engagement in future should\\
be undertaken before investigations to improve\\
compliance by the farmers and communities\\
4.\end{longtable} \par
 
\caption{\label{tab_0}}\end{figure}
 		 		\backmatter   			 
\subsection[{Acknowledgments}]{Acknowledgments}\par
We thank the Western Urban and Rural districts community stakeholders for their support and for allowing us to perform sampling; officers of the Ministry of Health and Sanitation, Ministry of Agriculture and Forestry; the Food and Agriculture Organization of the United Nations including Dr. Mohamed Vandi, Dr. Amadu Tejan Jalloh, Sorie Kamara, Bayoh Musa, Tommy Josephine; Minkailu, field teams including Mary, Satta, Zainabu, Leigh, Kadijja Kamara, Bintah Bah, Bangura, Ishmail, Lamin Bangura, Mohamed Jalloh, Hajja Bah, Saidu AB Turay, Christian Scoff, Fafia Bona, and Alhassan Jalloh for participating in community engagement and supporting sample collection. 
\subsection[{Conflict of Interest}]{Conflict of Interest}\par
The authors declare that there are no conflicts of interest regarding the publication of this paper.  			  			 
\subsection[{Funding Statement}]{Funding Statement}\par
This work was supported by the Food and Agriculture Organization of the United Nations, Sierra Leone. 			 			  				\begin{bibitemlist}{1}
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